Mycobacterium kansasii is a facultative pathogen which can cause a severe tuberculosis-like disease in man. About 10% of mycobacterial isolates from AIDS patients have been identified as M. kansasi. Because of the clinical importance of this nontuberculous mycobacterial species, we have prepared a lgt11 expression library. We have generated 4 M. kansasii monoclonal antibodies; one of these antibodies reacts specifically with a 35 kDa M. kansasii antigen. Recently, a bacteriophage has been isolated from the gene library that expresses recombinant M. kansasii immunoreactive proteins recognized by the monoclonal antibody. Phage DNA has been purified from these recombinant clones and the physical structure of the 35 kDa antigen gene has been analyzed. The recombinant M. kansasii protein which was overproduced in the pGEM expression system has been purified by electroelution and HPLC. In M. kansasii sensitized guinea pigs, this recombinant antigen elicits a large skin test reaction. Further studies are in progress to determine the specificity of this skin test response and to evaluate whether this antigen will be useful as a monospecific clinical reagent in the diagnosis of M. kansasii infections.